This invention relates to antisense therapy. More particularly, this invention relates to compositions and methods for enhancing the cellular uptake of antisense oligonucleotides.
New chemotherapeutic agents have been developed which are capable of modulating cellular and foreign gene expression. These agents, called antisense oligonucleotides, are single-stranded oligonucleotides which bind to a target nucleic acid molecules according to the Watson-Crick or Hoogsteen rule of base pairing, and in doing so, disrupt the function of the target by one of several mechanisms: by preventing the binding of factors required for normal transcription, splicing, or translation; by triggering the enzymatic destruction of RNA by RNase H, or by destroying the target via reactive groups attached directly to the antisense oligonucleotide. Thus, they have become widely used research tools for inhibiting gene expression sequence specifically, and are under investigation for possible use as therapeutic agents (see, e.g., Agrawal et al. (Proc. Natl. Acad. Sci. (USA) (1993) 90:3860-3884); Bayever et al. (1992) Antisense Res. Development 2: 109-110).
In order for antisense molecules to have therapeutic value, they must have the ability to enter a cell and contact target endogenous nucleic acids. Furthermore, they must be able to withstand the rigors of the highly nucleolytic environment of the cell.
Recent studies have shown that certain modifications to oligonucleotides, such as artificial internucleotide linkages, not only render the oligonucleotides resistant to nucleolytic degradation (see, e.g., Agrawal et al. (1988) Proc. Natl. Acad. Sci. (USA) 85: 7079-7083; Agrawal et al. (1989) Proc. Natl. Acad. Sci. (USA) 86: 7790-7794; Gao et al. (1990) Antimicrob. Agents Chem. 34:808; and Storey et al. (1991) Nucleic Acid Res. 19:4109), but also may increase cellular uptake of the oligonucleotide. For example, oligonucleotides with phosphorothioate or methylphosphonate internucleotide linkages have been found to bind to, and to be taken up by cells more readily than phosphodiester-linked oligonucleotides (Zhao et al. (1993) Antisense Res. Dev. 3:53-56).
Oligonucleotide uptake is saturable, sequence-independent, and temperature and energy dependent. While there is some evidence to suggest that such uptake may occur through a 80,000 dalton membrane protein (Loke et al. (1989) Proc. Natl. Acad. Sci. (USA) 86:3474; Yakubov et al. (1989) Proc. Natl. Acad. Sci. (USA) 86:6454), the gene for this protein has not yet been cloned or characterized. One study suggests internalization of the oligonucleotide is by a caveolar, potocytotic mechanism rather than by endocytosis (Zamecnick (1994) Proc. Natl. Acad. Sci. (USA) 91:3156). Whether oligonucleotides are internalized via a receptor-mediated endocytotic pathway, a pinocytic mechanism, or a combination of both remains poorly understood.
To improve on the cellular uptake of oligonucleotides, the oligonucleotides have been modified in ways other than those described above. For example, WO 9323570 discloses an oligonucleotide with improved cellular uptake having at least one nucleotide residue covalently linked at its 2' position with various molecules including an amino acid, polypeptide, protein, sugar, sugar phosphate, neurotransmitter, hormone, cyclodextrin, starch, steroid, or vitamin. Enhanced cellular uptake of biotinylated oligonucleotide in the presence of avidin has also been demonstrated (Partridge et al. (1991) FEBS Lett. 288:30-32).
In addition, phosphodiester-linked oligodeoxynucleotides have been introduced into cells by the pore-forming agent streptolysin O (Barry et al. (1993) Biotechniques 15:1016-1018), and a liposomal preparation including cationic lipid has been shown to enhance the cellular uptake of antisense molecules targeted to a portion of the human intercellular adhesion molecule (Bennett et al. (1992) Mol. Pharmacol. 41:1023-1033). phosphodiester-linked oligonucleotides bearing a 5'-cholesteryl modification show increased cellular uptake and biological effects (Krieg et al. (1993) Proc. Natl. Acad. Sci. (USA) 90:1048). Antibody-targeted liposomes have also been used to enhance the cellular uptake of oligonucleotides targeted to HLA class I molecules expressed by HIV-infected cells (Zelphati et al. (1993) Antisense Res. Dev. 3:323-338).
However, improved uptake Of modified and unmodified oligonucleotides both in vitro and in vivo is obviously desirable. There is therefore a need for improved compositions and methods for enhancing the cellular uptake of antisense oligonucleotides. Such enhancement would ultimately result in an increased efficacy of antisense oligonucleotides and a reduction in the dose administered. Ideally, such compositions and methods will also be useful for increasing the general solubility of oligonucleotides.